Ji-Hun Seo

Inducing Rapid Cellular Response on RGD-Binding Threaded Macromolecular Surfaces

The rapid response of integrin β1 molecules to an RGD peptide on a dynamic polyrotaxane surface was successfully induced. As a result, RGD peptides introduced on a highly dynamic cyclodextrin molecule enhanced the frequency of contact with specific integrin molecules on the cell membrane at the early stage of material-cell interactions.

Adsorption state of fibronectin on poly(dimethylsiloxane) surfaces with varied stiffness can dominate adhesion density of fibroblasts

The state of adsorbed fibronectin and the subsequent cell adhesion behavior on polydimethylsiloxane (PDMS) substrates with varied stiffness were investigated. The bulk elastic modulus as well as the macroscale and nanoscale surface repulsion forces on PDMS substrates with five different cross-linker concentrations (2.5, 5, 10, 20 and 40wt.%) were evaluated by using tensile and compression tests as well as atomic force microscopy (AFM) indentation. The PDMS substrate with 10wt.% cross-linker showed the maximum stiffness in the bulk elastic modulus and macroscale compression test. In contrast, PDMS substrates with 2.5 and 5wt.% cross-linker concentration showed the maximum stiffness in the nanoscale compression test, which indicates that the physical properties of the nanoscale outermost surface are different from the bulk and macroscale surface properties. The fibronectin-treated PDMS substrates showed almost the same amount of fibronectin adsorption. However, the outermost surface density of fibronectin was related to the macroscale surface stiffness, and the exposure of the cell-binding motif was related to the nanoscale surface stiffness. Moreover, the different adsorption state of fibronectin was further confirmed by quartz crystal microbalance-dissipation (QCM-D) monitoring. The adhesion behavior of NIH3T3 mouse fibroblasts was in turn related to the exposure of the cell-binding motif. These results suggest that the well-known differences in cell adhesion behavior on PDMS substrates with varied stiffness are primarily induced by different responses of fibronectin to the PDMS substrates.

Designing dynamic surfaces for regulation of biological responses

ABA block copolymers composed of highly methylated polyrotaxane and hydrophobic anchoring terminal segments containing 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (PMB) (OMe-PRX-PMB) were synthesized as a platform of molecularly dynamic biomaterials. A contact angle measurement indicated that polymer surfaces with higher molecular mobility factors (Mf) estimated from quartz crystal microbalance with dissipation (QCM-D) measurements showed more significant changes in hydrophilicity in response to an environmental change between air and water; the OMe-PRX-PMB surface showed the highest Mf among the prepared polymer surfaces. Fibrinogen adsorption and its conformational analysis estimated by QCM-D and enzyme-linked immunosorbent assay revealed that large amounts of fibrinogen adsorption occurred in a soft manner on the OMe-PRX-PMB surface and that the antibody binding to the C-terminus of the fibrinogen γ chains responsible for platelet adhesion and activation decreased as the Mf value increased. Furthermore, it was found that the OMe-PRX-PMB surface showed low platelet adhesion and high fibroblast adhesion, suggesting that molecular movement on biomaterial surfaces could be one of the key parameters in the regulation of a non-specific biological response.

The significance of hydrated surface molecular mobility in the control of the morphology of adhering fibroblasts

The effects of the hydrated molecular mobility and the surface free energy of polymer surfaces on fibronectin adsorption and fibroblast adhesion were investigated. ABA-type block copolymers composed of polyrotaxane (PRX) with different number of threaded α-cyclodextrin (α-CD), random copolymers with similar chemical composition to the PRX block copolymers, and conventional polymers were prepared to determine a wide range of hydrated molecular mobility (Mf) values estimated by quartz crystal microbalance-dissipation (QCM-D) measurements. Fibronectin adsorption was highly dependent on surface free energy, and high surface fibronectin density resulted in a large projected cell area on the polymer surfaces. However, the morphology of adhering fibroblasts was not explained by the surface free energy, but it was found to be strongly dependent on the Mf values of the polymer surfaces in aqueous media. These results emphasize the importance of Mf in the discussion of the elongated morphology of adhering fibroblasts on various polymer surfaces.

The effect of molecular mobility of supramolecular polymer surfaces on fibroblast adhesion

The effect of hydrated molecular mobility of polymer surfaces on cell adhesion behavior was investigated. ABA-type block copolymers composed of polyrotaxane (PRX) and hydrophobic anchoring terminal segments were synthesized as a platform of molecularly mobile surfaces. The result of QCM-D measurement in water revealed that the molecularly mobile PRX block copolymer surfaces were higher in hydrated molecular mobility than the corresponding random copolymer surfaces with similar content of hydrophobic methoxy groups. The number of adhering fibroblasts depended on the amount of fibronectin adsorbed from serum but was independent of the molecular mobility. However, the morphology of the adhering fibroblasts was strongly dependent on the extent of molecular mobility in water. These results indicate that molecular mobility on polymer surfaces is one of the significant considerations for regulating cellular responses.

Molecular logistics using cytocleavable polyrotaxanes for the reactivation of enzymes delivered in living cells

The intracellular delivery of enzymes is an essential methodology to extend their therapeutic application. Herein, we have developed dissociable supermolecule-enzyme polyelectrolyte complexes based on reduction-cleavable cationic polyrotaxanes (PRXs) for the reactivation of delivered enzymes. These PRXs are characterized by their supramolecular frameworks of a polymeric chain threading into cyclic molecules, which can form polyelectrolyte complexes with anionic enzymes while retaining their three dimensional structure, although their enzymatic activity is reduced. Upon the addition of a reductant, the PRXs dissociate into their constituent molecules and release the enzymes, resulting in a complete recovery of enzymatic activity. Under the intracellular environment, the PRX-based enzyme complexes showed the highest intracellular enzymatic activity and efficient activation of anticancer prodrugs to induce cytotoxic effects in comparison with the non-dissociable complexes and the commercial cell-penetrating peptide-based reagents. Thus, the intracellularly dissociable supermolecules are an attractive system for delivering therapeutic enzymes into living cells.

Directing Stem Cell Differentiation by Changing the Molecular Mobility of Supramolecular Surfaces

Polymer surfaces with a wide range of hydrated surface mobility are developed by a simple deposition method with supramolecular block copolymers. The morphologies of adhering stem cells are greatly dependent on the surface mobility of polymers, and this induces significant changes in the cytoskeletal signaling pathway to direct the downstream stem cell differentiation.

Development of anti-biofouling interface on hydroxyapatite surface by coating zwitterionic MPC polymer containing calcium-binding moieties to prevent oral bacterial adhesion.

UNLABELLED The purpose of the present study is to synthesize a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer capable of being immobilized on the tooth surface to prevent oral bacterial adhesion. The strategy is to develop an MPC-based polymer with Ca(2+)-binding moieties, i.e., phosphomonoester groups, for stronger binding with hydroxyapatite (HA) of the tooth surface. To this end, a 2-methacryloyloxyethyl phosphate (MOEP) monomer was synthesized and copolymerized with MPC by free radical polymerization. The coating efficiency of the synthesized polymer, MPC-ran-MOEP (abbreviated as PMP) with varied composition, onto a HA surface was estimated by means of contact angle measurement and X-ray photoelectron spectroscopy. The anti-biofouling nature of PMP-coated HA surfaces was estimated by analyzing protein adsorption, cell adhesion, and Streptococcus mutans adhesion. As a result, HA surface coated with a copolymer containing around 50% MPC (PMP50) showed the best performance in preventing protein adsorption and the downstream cell and bacterial adhesion. STATEMENT OF SIGNIFICANCE Preparation of anti-biofouling surface on the tooth enamel is the key technique to prevent dental and periodontal diseases, which are closely related with the biofilm formation that induced by the adsorption of salivary proteins and the adhesion of oral bacteria on the tooth surface. In this research, a PMP copolymer with an optimized ratio of zwitterionic and Ca(2+)-binding moieties could form a highly effective and robust anti-biofouling surface on HA surfaces by a simple coating method. The PMP-coated surface with high stability can provide a new strategy for an anti-adsorptive and anti-bacterial platform in dentistry and related fields.

A large mobility of hydrophilic molecules at the outmost layer controls the protein adsorption and adhering behavior with the actin fiber orientation of human umbilical vein endothelial cells (HUVEC)

Adhesion behaviors of human umbilical vein endothelial cells (HUVECs) are interestingly affected by the mobility of hydrophilic chains on the material surfaces. Surfaces with different molecular mobilities were prepared using ABA-type block copolymers consisting polyrotaxane (PRX) or poly(ethylene glycol) (PEG) central block (A block), and amphiphilic anchoring B blocks of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). Two different molecular mobilities of the PRX chains were designed by using normal α-cyclodextrin (α-CD) or α-CD whose hydroxyl groups were converted to methoxy groups in a given ratio to improve its molecular mobility (PRX–PMB and OMe-PRX–PMB). The surface mobility of these materials was assessed as the mobility factor (Mf), which is measured by quartz crystal microbalance with dissipation monitoring system. HUVECs adhered on OMe-PRX–PMB surface much more than PRX–PMB and PMB-block–PEG–block-PMB (PEG–PMB) surfaces. These different HUVEC adhesions were correlated with the density of cell-binding site of adsorbed fibronectin. In addition, the alignment of the actin cytoskeleton of adhered HUVECs was strongly suppressed on the PEG–PMB, PRX–PMB, and OMe-PRX–PMB in response to the increased Mf value. Remarkably, the HUVECs adhered on the OMe-PRX–PMB surface with much less actin organization. We concluded that not only the cell adhesion but also the cellular function are regulated by the molecular mobility of the outmost material surfaces.

Mobility of the Arg-Gly-Asp ligand on the outermost surface of biomaterials suppresses integrin-mediated mechanotransduction and subsequent cell functions

Mechanotransduction in the regulation of cellular responses has been previously studied using elastic hydrogels. Because cells interact only with the surface of biomaterials, we are focusing on the molecular mobility at the outermost surface of biomaterials. In this study, surfaces with the mobile Arg-Gly-Asp-Ser (RGDS) peptide have been constructed. Cell culture substrates were coated with ABA-type block copolymers composed of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) segments (A) and a polyrotaxane (PRX) unit with RGDS bound to α-cyclodextrin (B). Adhesion, morphological changes and actin filament formation of human umbilical vein endothelial cells were reduced on the surfaces containing mobile PRX-RGDS in comparison to the immobile RGDS surfaces constructed from random copolymers with RGDS side groups (Prop-andom-RGDS). In the neurite outgrowth assay using rat adrenal pheochromocytoma cells (PC12), only ∼20% of adherent PC12 cells had neurites on PRX-RGDS surfaces, but more than 50% did on the Random-RGDS surface. The beating colony of dimethyl-sulfoxide-treated mouse embryonic carcinoma cells (P19CL6) were found 10 and 14 days after induction on PRX-RGDS and Random-RGDS surfaces, respectively. After 22 days, the beating colony disappeared on PRX-RGDS surfaces, but many colonies remained on Random-RGDS surfaces. These data suggest that the molecular mobility of the cell-binding ligand on the outermost surface of materials effectively suppresses the actin filament formation and differentiation of these functional cell lines, and may be used as a culture substrate for immature stem cells or progenitor cells.